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Mulberry leaf has attracted much attention due to its excellent curative effect on diabetes and its complications, whether the combination of its effective components have protective and synergistic effect on diabetic nephropathy (DN) in vivo remain unclear.The aim of this study was to investigate the protective and synergistic effect of the combination (MAFA step by step method consisted of network pharmacological prediction, animal in vivo validation and metabolic mechanism research was used to construct the multi-component-target-pathway network of mulberry leaf against DN. Firstly, the potential components and mechanism of mulberry leaf against DN was explored by network pharmacology analysis. Secondly, DN animal model was established to validate the anti-DN activity of these potential compounds. Thirdly, the metabolomics of serum and urine samples from animal experiments was analyzed to explore the anti-DN mechanism of these potential compounds.The results of network pharmacology demonstrated that a total of 7 compounds detected in MA and MF exhibited anti-DN activity, their mechanism were strongly in connection with metabolic pathways, arachidonic acid metabolism, sphingolipid signaling pathway, etc. The results of animal experiment indicated that MAFMAF

In this study, a simple, sensitive, and rapid analytical method was developed by ultra-performance liquid chromatography (UPLC) coupled with tandem quadrupole mass spectrometry (MS) for the determination of nootkatone in normal and chronic kidney disease (CKD) rat plasma using clarithromycin as internal standard. After sample preparation by simple liquid–liquid extraction, chromatography was performed on an Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 mm particle size) using gradient elution with the mobile phase composed of acetonitrile and water acidified with 0.1% (v/v) formic acid. Detection was achieved by electrospray ionisation MS under the multiple selective reaction monitoring mode. The linear range was 0.01‒500 ng/mL with the square regression coefficient (r) of 0.9975. The lower limit of quantification was 0.01 ng/mL. The intra- and inter-day precision was under 5% and the stability accuracy was between 3.6 and 7.0%. The average recoveries from spiked plasma samples were >83% and matrix effect was over 81%. The developed method was successfully applied to the pharmacokinetic study of nootkatone in normal and CKD rats after an oral administration of 50 mg/kg nootkaone. The results showed the cmax and area under curve of nootkaone were greatly decreased, meanwhile Vd/F and t1/2 were markedly increased in CKD rats. The pharmacokinetic characteristics of nootkatone in rats were significantly altered in CKD rats.

Danshen (Salviae Miltiorrhizae Radix et Rhizoma, SMR) has been used as a traditional Chinese medicine in clinic for treatment of coronary heart diseases. Previous works have shown that the chronic renal failure (CRF) is closely related to changes of intestinal bacteria. The aim is to explore the interaction between active components of SMR and intestinal bacteria from normal and CRF rats. The changes of intestinal bacteria were evaluated among normal rats, CRF model rats and SMR-treated rats via 16S rRNA gene sequencing technology. UPLC-QTOF/MS was applied for the analysis and identification of metabolites. RESULTS: Results showed that the following intestinal bacteria varied significantly in CRF rats, including Mucispirillum, Kurthia, Clostridium, Blautia, Butyrivibrio, Shuttleworthia, Peptococcus, Ruminococcus, Bradyrhizobium, Methylobacterium, Azospirillum, Thalassospira, Methylophilus, Pseudomonas, peptostreptococcaceae and bacteroidales. The ethanol extract of SMR (DS) significantly regulated Shuttleworthia, peptostreptococcaceae and Pseudomonas, while the water extract (DSS) significantly affected Peptococcus, peptostreptococcaceae and Ruminococcus. Methylation, demethylation, dehydrogenation, hydrogenation and hydroxylation were the major metabolic transformation of tanshinones in vitro by intestinal bacteria. Glucuronidation, methylation and hydrogenation were the main metabolic transformation of salvianolic acids. These results showed that the bioactive components of SMR, including tanshinones and salvianolic acids, might exert the medical effect via regulation intestinal bacteria.

Diabetic nephropathy (DN) is a complication of diabetes that is caused by uncontrolled high blood sugar. It has been reported that Salvia miltiorrhiza (SM) possesses the ability to prevent kidney damage, although the mechanisms remain unclear. The study was to investigate whether and how SM improved DN injury via regulation of metabolome and the molecular mechanisms. In this study, SD rats were fed a high glucose / high fat diet accompanied by 0.5% glucose water. Three weeks later, the rats were given one intraperitoneal injection of 30 mg/kg STZ each day for three days for DN model. The biochemical indicators and metabolomics of plasma, urine and renal tissue were analyzed. Then the western blotting analysis of renal tissue and glomerular mesangial cells were investigated. The results showed that Salvia miltiorrhiza extracts improved the renal injury and regulation of abnormal glycolipid metabolism. The metabolites in serum, urine and renal tissues have been changed significantly. The involved metabolic pathways mainly include phospholipid, arachidonic acid, and pyrimidine metabolisms. Meanwhile, SM inhibited the relative expression levels of wnt4, β-catenin and TGF-β in renal tissue and high-glucose induced glomerular mesangial cells.

This study aimed to explore the effect of Salviae Miltiorrhizae Radix et Rhizoma, its stems and leaves on the diversity of intestinal microflora in rats with diabetic kidney injury. Diabetic rats model was established by feeding high glucose and high fat diet and 5% glucose solution with intraperitoneal injection of 30 mg·kg~(-1) streptozocin(STZ). The rats were randomly divided into normal group, model group, irbesartan control group, Huangkui Capsules control group, as well as low, middle and high dose groups of Sal-viae Miltiorrhizae Radix et Rhizoma, its stems and leaves. After administration for 2 weeks, 16 S rRNA technique was used to analyze the diversity of intestinal microflora in the feces of each group. The results showed rats in the model group developed renal tubular epithelial vacuole degeneration and a large amount of inflammatory cell infiltration in the renal interstitium. A small amount of inflammatory cell infiltration was seen in each administration group. The kidney structure of rats in irbesartan group, Huangkui Capsules group, high-dose group of Salviae Miltiorrhizae Radix et Rhizoma and its stem water extract, as well as high dose group of Salviae Miltiorrhizae Radix et Rhizoma and its stem ethnol extract group was close to the normal group. The diversity and structure of intestinal flora in the model group were significantly different from those in the normal group. Each administration group improved the fecal flora diversity in rats with diabetic kidney injury to a certain extent, especially the high dose of Salviae Miltiorrhizae Radix et Rhizoma and its stems water extract. Different flora were found in feces of diabetic nephropathy model rats on class, order, family and genus levels. On families and genera levels, the relative abundance of Bifidobacterium, Turicibacter, Peptostreptococcaceae, Desulfovibrio, and SMB53 showed an upward trend in model group, but that of Lactobacillus, Clostridium, Rikenella, Rumen fungi showed a downward trend. The administration groups can improve the relative abundance of the above intestinal flora in the model rats to a normal-like level. The results of this study provide a reference for resource utilization and further development of Salviae Miltiorrhizae Radix et Rhizoma.

Ethnopharmacological relevance The flower of Abelmoschus manihot (Linn.) Medicus ( A. manihot), as a traditional Chinese Herbal medicine, was used widely in China with efficacy of inducing diuresis for treating strangurtia, and subdhing swelling and detoxicating. It has been reported that Huangkui capsule, prepared by the extract of the flower of A. manihot , can reduce the content of urinary protein, serum creatinine and serum urea nitrogen in nephropathy rats and processes renoprotective activity, while the action mechanism need to illuminate deeply. Aims of the study In this study, we investigated the protection effect of Huangkui capsule on tubulointerstitial fibrosis in chronic renal failure (CRF) rats and its mechanism against high glucose-induced epithelial to mesenchymal transition (EMT) in renal tubular epithelial cells (HK-2) of its bioactive components. Materials and methods The animals were divided into normal group, CRF model group and Huangkui capsule-treated group. Hematoxylin eosin (HE) staining and Masson staining were applied to observe pathological changes in renal tissue of different groups. Biochemical indicators including serum urea nitrogen (BUN), urine protein (UP) and serum creatinine (Scr) were measured according to the manufacturer's instructions of kits. HK-2 cell damaged model was established to access the protection effect and action mechanism of five main flavonoids from Huangkui capsule. The experimental cells were divided into eight groups: control group, model group, positive drug group and five main flavonoids treated groups. The dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to determine the reactive oxygen species (ROS) in different groups. Western blot was applied to analyze the expression of pathogenesis-related proteins in different groups. Results The results stated that Huangkui capsule significantly inhibited the elevation of Scr, BUN, UP, the expression of α-smooth muscle actin (α-SMA), phosphorylation-extracellular signal-regulated kinase (p-ERK1/2), NADPH Oxidase 1, NADPH Oxidase 2 and NADPH Oxidase 4 in adenine-induced CRF rats. The main bioactive components of quercetin (QT), hyperoside (HY), isoquercitrin (IQT), gossypetin-8- O - β -D-glucuronide (GG) and quercetin-3′- O -glucoside (QG) at the dosage of 100 µM, like NADPH oxidase inhibitor diphenyleneiodonium, exhibited a significant effect on inhibiting the expression of α-SMA, p-ERK1/2, NADPH Oxidase 1, NADPH Oxidase 2 and NADPH Oxidase 4 in high glucose-induced HK-2 cells, especially GG. Conclusions These results demonstrated that Huangkui capsule and the flavonoids components prevent tubulointerstitial fibrosis in CRF rat involvement in the action mechanism of inhibiting NADPH oxidase/ROS/ERK pathway.

Rehmannia glutinosa Libosch (RG), is officially listed in the Chinese Pharmacopoeia and is widely used in China. The leaves of RG (LR) is an important vegetative organ of the plant. At present, the total glycosides of RG (TLR) were extracted from RG, and developed a national second class of new drugs to the Dihuangye total glycoside capsule (DTG). Additionally, DTG has the effect of nourishing yin and tonifying kidney, promoting blood circulation and blood cooling, and applicable to chronic glomerulonephritis mild to Qi and Yin Deficiency. Moreover, diabetic nephropathy (DN) rats model was induced by intraperitoneal injection of a small dose of streptozotocin (45 mg/kg) and high-fat diet and plus 5% glucose drinking water. Over 15 days, after oral administration TLR and DTG in DN rats, samples from serum, urine and kidney were collected for biochemical indicators measurements, pathological analysis, western blotting and metabolomics. Therefore, the analytical results of biochemical indicators, histopathological observations and western blotting showed that TLR and DTG exhibited a significant effect in renal protection. And 27 endogenous metabolites (12 in serum and 15 in urine) could be tentatively identified in the process of DN in rats using metabolomics method. Those endogenous metabolites were chiefly involved in sphingolipid metabolism; pentose, glucuronate interconversion; terpenoid backbone biosynthesis; purine metabolism and retinol metabolism. After drug intervention, these endogenous metabolites turned back to normal level some extent (P < 0.05). Furthermore, TLR and DTG prevent high glucose-induced glomerular mesangial cells (GMCs) by inhibiting TGF-β1 and Wnt/β-catenin signaling pathway, providing a powerful supports to develop a new therapeutic agent for DN. This study paved the way for further exploration of the pathogenesis of DN, early diagnosis and the evaluation of curative effect.

Abelmoschus manihot (L.) Medic., a folk herbal medicine in China, is a flowering plant belonging to Abelmoschus L. genus and Malvaceae family, which has been reported with an antidepressant activity. The study was designed to isolate flavonoids from Abelmoschus manihot corolla and explore the action mechanism of antidepressant activities. The flavonoids were isolated and purified by D101 macroporous resin column, polyamide column and Sephadex LH-20 sequentially and identified as myricetin-3-O-β-D-glucoside (1), gossypetin-8-O-β-D-glucuronide (2, G-8-G), gossypetin-3'-O-β-D-glucoside (3), quercetin-3'-glucoside (4, Q-3-G), isoquercitrin (5, IQT), hyperoside (6, HY), myricetin (7), quercetin (8, QT). Compounds 2, 4, 5, 6 and 8 (15, 30 and 60 mg·kg−1) were orally administered to mice and the reaction was observed in tail suspension test (TST) and forced swimming test (FST). Western blot analysis was used in determination of the protein expressions of brain-derived neurotrophic factor (BDNF), tyrosine receptor kinase B (TrkB) and phosphorylation eukaryotic elongation factor 2 (p-eEF2). The results revealed that only Q-3-G and G-8-G (15, 30, 60 mg ·kg−1) significantly reduced the immobility time in FST and TST. Furthermore, Q-3-G and G-8-G remarkably increased the expression of BDNF and TrkB, and decreased the expression of p-eEF2. These results suggest that Q-3-G and G-8-G had an obvious antidepressant activity via up-regulation of BDNF expression. The new observation will provide a new direction in the development of antidepressant in the treatment of major depressive disorder (MDD).

The evaluation of efficacy and safety should be paralleled with the assessment of comprehensive pharmacokinetic (PK) properties for a drug candidate, and a robust bioanalytical method is a prerequisite for obtaining PK information. α-Hederin is reported to have various in vitro and in vivo activities; however, very little is known about its PK and metabolic characteristics. In this study, we have developed an efficient LC-ESI(−)-MS/MS assay for α-hederin and its sapogenin hederagenin in rat plasma. Sample cleanup involved methanol precipitation for identification analysis and liquid–liquid extraction with ethyl acetate for quantification assay. LC analysis was performed under reversed-phase conditions in the modified “pulse gradient elution” mode. Analyte identification and quantification were conducted using multiple reaction monitoring (MRM) mode with euscaphic acid as the internal standard. Under these conditions, deglycosylated metabolites and their sulfate conjugates were detected; however, hederagenin was not detected in rat plasma samples after both oral and intravenous treatments. The mean plasma clearance (CL), volume of distribution (VSS) and elimination half-life (t1/2) of α-hederin were 0.24 L h−1 kg−1, 0.25 L kg−1 and 2.67 h, respectively. The oral bioavailability (F) of α-hederin in rats was about 0.14%, which might result from the poor intestinal absorption and/or extensive biliary excretion. It is hoped that this validated method will be useful for future PK studies of α-hederin.

Ethnopharmacological relevance Chronic renal failure (CRF) is defined as a progressive and irreversible loss of renal function and associated with inflammation and oxidative stress. Salvia miltiorrhiza (SM) is an important Chinese herb used in traditional Chinese medicine for treating cardiovascular diseases. The previous studies showed the SM exhibited significant protective effects on CRF. In this present study, the metabolic profiling changes and action mechanism of SM on CRF were explored. Aims of the study The aims of this study were to illustrate the metabolic profiling changes of adenine induced CRF and analyze the protective effects and action mechanisms of SM ethanol extract (SMEE) and water extract (SMWE). Materials and methods The animals were divided into normal group, CRF model group, Huangkui capsule-treated group, SMEE-treated group and SMWE-treated group. The UPLC−QTOFMS coupled with multivariate statistical methods were used to explore the changes of metabolic profile in plasma, urine and renal tissue from CRF rats simultaneously after treatment with SMEE and SMWE. Hematoxylin eosin (HE) staining and Masson staining were applied to observe pathological changes in renal tissue. Biochemical indicators including serum urea nitrogen (BUN), urine protein (UP) and serum creatinine (Scr) were measured according to the manufacturer's instructions of kits. Furthermore, HK-2 cell damaged model induced by ISF was established to access the protective effects and action mechanism. The dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to determine the reactive oxygen species (ROS) and Western blot was applied to analyze the expression of pathogenesis-related proteins in different groups. Results The results showed that the ethanol extract (SMEE) and water extract (SMWE) of SM significantly inhibited the elevation of serum creatinine (Scr), blood urea nitrogen (BUN), urine protein (UP) and indoxyl sulfate (ISF) in adenine-induced CRF rats, especially SMEE exhibited more significant effects. Moreover, SM extracts obviously improved the symptoms of glomerular and tubular atrophy, focal calcium deposits, interstitial fibrosis, interstitial inflammation, and renal tissues. By metabolomics analysis, fifty-nine metabolites (thirteen in plasma, twenty-seven in urine and nineteen in kidney tissue) were up-regulated or down-regulated and contributed to CRF progress. After treatment of SM extracts, the altered metabolites were restored back to normal level. These potential biomarkers underpinning the metabolic pathways are including phenylalanine metabolism, pyrimidine metabolism, purine metabolism and tryptophan metabolism. Furthermore, SM extracts prevent epithelial-mesenchymal transition (EMT) of human renal tubular epithelial (HK-2) cell by inhibiting NADPH oxidase/ROS/ERK and TGF-β/Smad signaling pathways. Conclusions SMEE and SMWE can significantly alleviate adenine-induced CRF via regulation of the metabolic profiling and modulation of NADPH oxidase/ROS/ERK and TGF-β/Smad signaling pathways, which provided important supports for the development of protective agent of SM for CRF.

This paper investigated the diversity of the silkworm excrement bacterial communities in different ages before and after drying, aiming to clarify the differences of bacterial communities in composition and bacterial abundance and the influences of drying treatment, and provide scientific basis for the efficacy of scientific connotation and utilization of silkworm excrement. High-throughput sequencing technique was used to measure the sequence of 16S rDNA-V4 variable region of bacteria in silkworm excrement. QIIME, Mothur and PICRUSt software programs were employed to sort and calculate the number of sequences and operational taxonomic units (OTUs) for each sample. Thereafter, the abundance, distribution, alpha diversity index of species, beta diversity and bacterial communities diversity among different sample groups and predicted the bacterial gene functions were analyzed. In this study, the numbers of effective sequences for six samples were 259 250; the rarefaction curves showed a sufficient sequencing depth, and the number of OTUs was close to saturation. The bacteria in silkworm excrement belonged to the following five phylums: Proteobacteria (89.3%), Actinobacteria (5.0%), Firmicutes (4.4%), Bacteroidetes (1.1%) and Cyanobacteria (0.2%). The dominant specie was Cyanobacteria of the total bacteria identified, respectively. The abundances and diversities of the silkworm excrement bacterial communities have been reduced after drying treatment, especially the silkworm excrement of the fifth instar. PICRUSt analysis was performed to show that abundance of the functional genes such as membrane transport, carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleotide metabolism, cellular processes and signaling were relatively high. The result showed that the drying treatment could decreased the species and numbers of pathogenic bacteria in silkworm excrement obviously and improve the quality of medicinal materials. Compared with the lower ages, silkworm excrement of fifth instar seems like to be more suitable for use in medicine. Illumina MiSeq high-throughput sequencing system provides a more accurate and scientific data resource for the study of bacteria in silkworm excrement.

Euscaphic acid, a triterpene acid, exists ubiquitously in medicinal plants and demonstrates various pharmacological activities. This active compound is often used as a marker compound for quality control. Hitherto, the pharmacokinetic (PK) information is relatively scarce; therefore, it remains open to question whether the euscaphic acid reaches the target sites in the body at concentrations high enough for the claimed biological effects. A robust analytical method is prerequisite for obtaining enough PK information of euscaphic acid, which is useful for interpreting its pharmacological effects. In this study, we developed and validated a rapid liquid chromatographic tandem mass spectrometric (LC-MS/MS) method for the measurement of euscaphic acid in rat plasma. The rat plasma samples were precipitated with acetonitrile and the resulting supernatants were separated by a 4 min pulse gradient method on a Synergi Fusion-RP C18 column (4 μm, 2.0 mm i.d. × 50 mm). Ursolic acid was used as an internal standard for quantification of euscaphic acid. Deprotonated euscaphic acid and its internal standard were generated in negative electrospray ionization (ESI) mode and their precursor-to-product ion pairs (m/z 487.4→469.3 and 455.5→455.4, respectively) were used for measurement. Notably, the commonly used concentration of formic acid (HCOOH; 1‰ and 5‰, v/v) in the mobile phase seriously suppressed the signal intensity, but this mobile phase additive at a much lower concentration level (0.1‰ and 0.2‰) could overcome the matrix effects and therefore increase the sensitivity of MS detection of euscaphic acid. The newly developed bioanalytical assay possessed favorable accuracy and precision with a lower limit of quantification of 2.0 ng mL−1 and was successfully applied to PK studies in rats. The experimental strategies presented herein may be helpful for measurement of other triterpene acids in biological matrices.

Abelmoschus manihot was rich in flavonoids, which has been reported the activity on protecting angiocarpy and improving renal function. This study aimed to explore the action mechanism of five flavonoids from A. manihot on how to ameliorating insulin resistance through the regulation of the glucose and expression of PPARγ, C/EBPα, SREBP-1, resistin, visfatin, adiponectin in 3T3-L1 adipocytes. After the 3T3-L1 preadipocytes were differentiated into mature adipocytes, insulin resistance model was built. Insulin resistance adipocytes were treated with 5, 100 μmol•L⁻¹ quercetin, isoquercitrin, hyperoside, quercitrin-3'-O-glucoside, gossypetin-8-O-β-glucoside. The glucose was indirectly determined by BCA kit. The mRNA expression levels of PPARγ, C/EBPα, SREBP-1, resistin, visfatin, adiponectin were detected by real-time quantitative PCR. Results showed that five flavonoids at 5 μmol•L⁻¹ could accelerate preadipocytes proliferation and inhibit that at 100 μmol•L⁻¹ Compared with the normal group, glucose uptake reduced significantly in model group (P

Alpinia oxyphylla (Zingiberaceae) is a well-known medicinal plant. Its fruit ("Yi-Zhi-Ren" in Chinese) is used as an anti-diuretic and traditionally used for the treatment of enuresis and reduce urination. Chronic kidney disease (CKD) is a disease with the characteristic of the slowly loss of kidney function and has a prevalence of up to 7-10% in adults. Recent advances in its etiology and pathogenesis are providing more speculative hypotheses focused on integral systems. Using a UPLC-QTOF-MS/MS-based metabolomic platform, we explored the changes of metabolic profiling in plasma/urine simultaneously between chronic kidney disease (CKD) induced from adenine excess and the protective effects of A. oxyphylla extract (AOE). The total twenty-one metabolites (twelve in urine and nine in plasma), up-regulated or down-regulated, were identified and contributed to CKD progress. Among these biomarkers, agmatine, CAMP, 7-methylguanine, hippuric acid, indoxyl sulfate, asparagines, kynurenic acid and p-cresol sulfate were restored back to the control-like level after the treatment of AOE (p

Background Galangin (3,5,7-trihydroxyflavone) is present in high concentrations in herbal medicine such as Alpinia officinarum Hance. Galangin shows multifaceted in vitro and in vivo biological activities. The number and position of hydroxyl groups in this molecule play an important role in these biological activities. However, these hydroxyl groups undergo glucuronidation and sulfation in in vitro assay system. However, the systemic exposure to galangin after dosing in animals and/or humans remains largely unknown. Thus it is not clear whether the galangin exists in the body at concentrations high enough for the biological effects. Furthermore, the metabolite identification and the corresponding plasma pharmacokinetics need to be characterized. Results Two LC-MS/MS methods were developed and validated and successfully applied to analyze the parent drug molecules and aglycones liberated from plasma samples via β-glucuronidase hydrolysis. Our major findings were as follows: (1) The routes of administration showed significant influences on the systemic exposure of galangin and its metabolites. (2) Galangin was preferentially glucuronidated after p.o. dosing but sulfated after i.v. medication. (3) Kaempferol conjugates were detected demonstrating that oxidation reaction occurred; however, both glucuronidation and sulfation were more efficient. (4) Oral bioavailability of free parent galangin was very low. Conclusions Systemic exposure to galangin and its metabolites was different in rat plasma between oral and intravenous administration. Further research is needed to characterize the structures of galangin conjugates and to evaluate the biological activities of these metabolites.

Rehmannia glutinosa Libosch (RG), is officially listed in the Chinese Pharmacopoeia and is widely used in China. In this paper, a sensitive and rapid ultra-performance liquid chromatography-mass spectrometry method including multiple-reaction monitoring mode was developed and applied to study the pharmacokinetic effect of acteoside from total glycoside extracted from the leaves of Rehmannia (TLR) and Dihuangye total glycoside capsule (DTG) in normal and diabetic nephropathy rats. The diabetic nephropathy rat model was induced by intraperitoneal injection of a small dose of streptozotocin and high-fat diet and plus 5% glucose drinking water. Samples of plasma of rats were obtained at different times after rats were administered TLR (7.2 g/kg) and DTG (360 mg/kg). After deproteinization by acetonitrile, the concentrations of acteoside in rats at different time points were detected by UPLC-TQ-MS method and pharmacokinetics parameters were calculated using DAS 3.2.8 software. A good linearity of acteoside was shown in the range of 8.51-3404.8 ng/m L (r2 = 0.9987). The mean extraction recovery of analyte was in the range of 63.55-79.49%, and the intra- and inter-day RSD values were